naphthoquinones of the spinochrome class: occurrence ... · naphthoquinones of the spinochrome...
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Naphthoquinone
aDepartment of Food Science, University of
ZealandbLaboratory of the Chemistry of Natural Qu
Institute of Bioorganic Chemistry, Far Easter
Prospect 100 let Vladivostoku 159/2,
[email protected] of Biochemistry, University of
ZealanddDepartment of Microbiology and Immuno
Dunedin 9054, New Zealand
Cite this: RSC Adv., 2018, 8, 32637
Received 5th June 2018Accepted 17th August 2018
DOI: 10.1039/c8ra04777d
rsc.li/rsc-advances
This journal is © The Royal Society of C
s of the spinochrome class:occurrence, isolation, biosynthesis and biomedicalapplications
Yakun Hou, a Elena A. Vasileva,b Alan Carne,c Michelle McConnell,d Alaa El-Din A.Bekhit a and Natalia P. Mishchenko*b
Quinones are widespread in nature and have been found in plants, fungi and bacteria, as well as in members
of the animal kingdom. More than forty closely related naphthoquinones have been found in echinoderms,
mainly in sea urchins but occasionally in brittle stars, sea stars and starfish. This review aims to examine
controversial issues on the chemistry, biosynthesis, functions, stability and application aspects of the
spinochrome class, a prominent group of secondary metabolites found in sea urchins. The emphasis of
this review is on the isolation and structure of these compounds, together with evaluation of their
relevant biological activities, source organisms, the location of origin and methods used for isolation and
identification. In addition, the studies of their biosynthesis and ecological function, stability and chemical
synthesis have been highlighted. This review aims to establish a focus for future spinochrome research
and its potential for benefiting human health and well-being.
1. Introduction
Rational use of biological resources of the aquatic environmentis a realistic scientic and practical goal since they represent70% of the organisms on earth. Marine hydrobionts, such as seaurchins, and specically their gonads, are a valuable renewablefood resource. At the same time, they can serve as a uniquesource of various natural compounds, which can be the basisfor the creation of various biomaterials,1,2 effective medicinaland parapharmaceutical preparations,3 as well as functionalfood products.3–5 Many species of regular sea urchins arecommercially harvested because their gonads are consumed inmany countries of Asia, the Mediterranean and North America.Aer the removal of the gonads, large amounts of sea urchinshells are le as waste. This shell material is rich in bioactivequinonoid pigments, principally spinochromes.4–6 Althoughthis class of compounds has been known for over 100 years,7,8
there is still some controversy in relation to their structures,stability, biosynthesis and ecological functions. In this review,a critical evaluation of existing data on spinochrome structures,
Otago, PO Box 56, Dunedin 9054, New
inonoid Compounds, G.B. Elyakov Pacic
n Branch of Russian Academy of Sciences,
690022, Vladivostok, Russia. E-mail:
Otago, PO Box 56, Dunedin 9054, New
logy, University of Otago, PO Box 56,
hemistry 2018
biosynthesis, distribution, isolation and identication tech-niques, stability, synthesis and biomedical applications ispresented.
2. The structure and nomenclature ofspinochromes
Spinochromes are polyhydroxylated derivatives of either juglone(5-hydroxy-1,4-naphthoquinone) or naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) that are substituted withvarious functional groups such as ethyl, acetyl, methoxyl, andamino groups. The rst discovered compound of this class,a red pigment in the perivisceral (coelomic) uid of Echinusesculentus was called ‘echinochrome’, without knowing itschemical composition.9 The structure of this ‘echinochrome’was established later by Kuhn andWallenfels (1940)10 as 6-ethyl-2,3,7-trihydroxynaphthazarin and it was named echinochromeA (Table 1, structure 1). Subsequently, a number of pigmentcompounds were isolated from the shells and spines of varioussea urchin species, which were named spinochromes todistinguish them from the echinochrome A found in thecoelomic uid, ovaries and internal organs of sea urchins.7
However, it was found later that echinochrome A is also a majorpigment component of the shells and spines of many seaurchins, so it was assigned to the class of spinochromes, but theoriginal name echinochrome has remained.18
New spinochromes were named based on the rst letter ofthe name of the sea urchin species that they were isolated from,as for example, spinochrome P was rst isolated from
RSC Adv., 2018, 8, 32637–32650 | 32637
Tab
le1
Structuresofkn
ownsp
inoch
romes
No.
Structureeluc
idation
R2
R3
R5
R6
R7
R8
Molecular
form
ula
Molecular
mass
Trivial
nam
eReferen
ces
16-Ethyl-2,3,5,7,8-pen
tahyd
roxy-1,4-nap
hthoq
uinon
eOH
OH
OH
C2H
5OH
OH
C12H
9O7
265
Ech
inochromeA
72
2-Acetyl-3
,5,6,8-tetrahyd
oxy-1,4-nap
hthoq
uinon
eCOCH
3OH
OH
OH
HOH
C12H
7O7
263
SpinochromeA
73
2,3,5,7-Tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
OH
OH
HOH
HC10H
6O6
221
SpinochromeB
74
2-Acetyl-3
,5,6,7,8-pen
tahyd
roxy-1,4-nap
hthoq
uinon
eCOCH
3OH
OH
OH
OH
OH
C12H
7O8
279
SpinochromeC
75
2,3,5,7,8-Pe
ntahyd
roxy-1,4-nap
hthoq
uinon
eOH
OH
OH
HOH
OH
C10H
6O7
237
SpinochromeD
76
2,3,5,6,7,8-Hexah
ydroxy-1,4-nap
hthoq
uinon
eOH
OH
OH
OH
OH
OH
C10H
6O8
252
SpinochromeE
77
6-Ethyl-2,5-dihyd
roxy-1,4-nap
hthoq
uinon
eOH
HOH
C2H
5H
HC12H
10O
421
811
86-Acetyl-2
,5,7-trihyd
roxy-1,4-nap
hthoq
uinon
eOH
HOH
COCH
3OH
HC12H
8O6
248
119
6-Ethyl-2,3,5,7-tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
OH
OH
C2H
5OH
HC12H
10O
625
011
106-Acetyl-2
,3,5,7-tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
OH
OH
COCH
3OH
HC12H
8O7
264
1111
3-Acetyl-2
,5,6,7-tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
COCH
3OH
OH
OH
HC12O7H
826
411
122,5,8-Tryihyd
roxy-1,4-nap
hthoq
uinon
eOH
HOH
HH
OH
C10O5H
620
6Nap
hthop
urpu
rin
1113
3-Acetyl-2
,5,8-trihyd
roxy-1,4-nap
hthoq
uinon
eOH
COCH
3OH
HH
OH
C12O6H
824
811
146-Ethyl-2,5,8-trihyd
roxy-1,4-nap
hthoq
uinon
eOH
HOH
C2H
5H
OH
C12O5H
10
234
1115
6-Acetyl-2
,5,8-trihyd
roxy-1,4-nap
hthoq
uinon
eOH
HOH
COCH
3H
OH
C12O6H
824
811
163-Ethyl-2,5,7,8-tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
C2H
5OH
HOH
OH
C12H
10O
625
011
172,5,7,8-Tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
HOH
HOH
OH
C10H
6O6
222
Mom
pain
1118
2-Hyd
roxy-2
0 -methyl-2
0 H-pyran
o[2,3-b]nap
hthazarin
OH
OH
C4-unit
OH
12
32638 | RSC Adv., 2018, 8, 32637–32650 This journal is © The Royal Society of Chemistry 2018
RSC Advances Review
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Tab
le1
(Contd.)
No.
Structureeluc
idation
R2
R3
R5
R6
R7
R8
Molecular
form
ula
Molecular
mass
Trivial
nam
eReferen
ces
193-Acetyl-2
,7-dihyd
roxy-6-m
ethyl-1,4-nap
hthoq
uinon
eOH
CH
3CO
OH
CH
3OH
OH
C13H
11O
727
812
206-Ethyl-3,5,6,8-tetrahyd
roxy-2-m
ethoxy-1,4-nap
hthoq
uinon
eOCH
3OH
OH
C2H
5OH
OH
C13H
12O
728
013
216-ethyl-2,5,6,8-tetrahyd
roxy-3-m
ethoxy-1,4-nap
hthoq
uinon
eOH
OCH
3OH
C2H
5OH
OH
C13H
12O
728
013
223,5,6,7,8-Pe
ntahyd
roxy-2-m
ethoxy-1,4-nap
hthoq
uinon
eOCH
3OH
OH
OH
OH
OH
C11H
8O8
268
Nam
akochrome
1428
2,3,5,8-Tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
OH
OH
HH
OH
C10H
6O6
222
Spinazarin
1529
6-Ethyl-2,3,5,8-tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
OH
OH
C2H
5H
OH
C12H
10O
625
0Ethylsp
inazarin
1530
3-Amino-6-ethyl-2,5,6,8-tetrahyd
roxy-1,4-nap
hthoq
uinon
eOH
NH
2OH
C2H
5OH
HC12H
11N
O6
265
Ech
inam
ineA
1631
2-Amino-6-ethyl-3,5,7,8-tetrahyd
roxy-1,4-nap
hthoq
uinon
eNH
2OH
OH
C2H
5OH
OH
C12H
11N
O6
265
Ech
inam
ineB
1632
2-Amino-3,5,6,7,8-pe
ntahyd
roxy-1,4-nap
hthoq
uinon
eNH
2OH
OH
OH
OH
OH
C10H
7NO7
253
Spinam
ineE
17
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Paracentrotus lividus. Spinochromes extracted from several seaurchin species have been named as spinochromes A, F, C, Mand H as well as others that can be found in the literature.18 Itwas later shown that many quinones previously designated withdifferent letters are in fact the same compound. Goodwin andSrisukh (1950)19 revised the nomenclature of all spinochromesknown at that time and proposed naming them in alphabeticorder, as spinochromes A (2), B (3), C (4), D (5) and E (6), asindicated for the structures summarised in Table 1. Aside fromthe original naming of these six most commonly found spino-chromes, polyhydroxylated naphthoquinone (PHNQ) pigmentsare now generally named as substituted juglones ornaphthazarins.18
Anderson et al. (1969)7 summarized the data on the distri-bution of spinochromes in almost 60 species of sea urchins andestablished that echinochrome A and the spinochromes A–E arethe most common pigments found in sea urchins. Thecomposition of different PHNQs is specic for different seaurchin species, and in addition to the six most common spi-nochromes, some species may also contain specic structuralanalogues. For example, for the sea urchins Echinotrix calamarisand Echinothrix diadema from the Hawaiian coast, nine quino-noid pigments (7–16) that are derivatives of either juglone ornaphthazarin were isolated along with mompain (17),commonly found in fungi,20 and 2-hydroxy-20-methyl-20H-pyr-ano[3,2-b]-naphthazarin (18), the only spinochrome known tohave a C4 unit attached to the naphthoquinone ring structure.11
The compound 3-acetyl-2,7-dihydroxy-6-methylnaphthazarin(19) was rst isolated from the shells and spines of the seaurchin Strongylocentrotus nudus (now known as Mesocentrotusnudus).21
Two monomethyl ethers of echinochrome A (20, 21) havebeen isolated from the tropical sea urchin Diadema antillarum.13
Methylated spinochromes previously had only been isolatedfrom other echinoderms, such as asteroids, holothuroids andophiuroids.13 Subsequently, these compounds were also foundin other Diadema species such as D. setosum and D. savignyi22
and very recently in the north pacic sea urchin Strong-ylocentrotus droebachiensis.23 Furthermore, in S. droebachiensisand its related species S. polyacanthus, Vasileva et al. (2017)23
reported two novel compounds by using HPLC-DAD-MS: mono-and dimethoxy derivatives of spinochrome E (22) that have notbeen found in other sea urchins, reported previously only inasteroids and holothuroids.
From heart urchin Spatangus purpureus, the rst spino-chrome dimers – ethylidene-3,30-bis(2,6,7-trihydroxynaphtha-zarin) (23) and its anhydro derivative (24) were isolated.13
Later, Utkina et al. (1976)24 working with Strongylocentrotusintermedius and Kol'tsova et al. (1978)25 working with S. droe-bachiensis, isolated binaphthoquinones with the same molec-ular mass. Based on different melting point and detailed 13C-NMR spectra analysis it was established that the ethylidenebridge binds the phenyl but not quinonoid rings of the twonaphthazarin fragments of the isolated binaphthoquinones, sotheir structures were elucidated as ethylidene-6,60-bis(2,3,7-trihydroxynaphthazarin) (25) and 7,70-anhydroethylidene-6,60-bis(2,3,7-trihydroxynaphthazarin) (26). Recently, binaphthoquin-
32640 | RSC Adv., 2018, 8, 32637–32650
ones with characteristics similar to compounds 23 and 25 and itsanhydrous derivative with characteristics similar to compounds 24and 26were isolated from the tropical sea urchin Astropyga radiata,and their structures were analysed using 2D-NMR procedures thatwere not available earlier.26 It was established that thesecompounds predominantly exist as ethylidene-3,30-bis(2,6,7-trihydroxynaphthazarin) (23) and 7,70-anhydroethylidene-6,60-bis(2,3,7-trihydroxynaphthazarin) (26), respectively.26 In additionto these two binaphthoquinones from sand dollar Scaphechinusmirabilis, the isomer of 26 was isolated, which was the rstunsymmetrical binaphthoquinone mirabiquinone (7,50-anhy-droethylidene-6,60-bis(2,3,7-trihydroxynaphthazarin) (27).27
Four novel compounds, spinazarin (28) and ethylspinazarin(29) that previously were not considered to be natural pigments,and echinamines A (30) and B (31), the rst representatives ofaminated spinochromes, were all isolated from S. mirabilis.15,16
A Chinese group using UPLC identied two new aminatedquinonoid pigments, aminopentahydroxynaphthoquinone andacetylaminotrihydroxynaphthoquinone, in the sea urchin Mes-ocentrotus nudus.5 Later, the amino-pentahydroxynaphthoquinone was isolated from the seaurchins M. nudus and Strongylocentrotus pallidus, and thestructure of the compound was elucidated to be 2-amino-3,5,6,7,8-pentahydroxy-1,4-naphthoquinone using 1D 1H- and13C-NMR and 2DNMR procedures, and was named spinamine E(32).17
Powell et al. (2014)28 using LC-MS discovered sulphatedspinochromes B and E and their structures were deduced basedon the measurement of their accurate masses. Sulphated spi-nochromes had not been reported previously in sea urchins andthe role of sulphated PHNQ pigments is still unknown. Furtherwork is required to conrm their structures and characteristics.
3. Biosynthesis of spinochromes
In contrast to plant and fungal quinones, biogenesis of animal-derived naphthoquinones is not well investigated. The pioneerstudy of spinochrome biosynthesis was performed by Salaqueet al. (1967)29 using the adult Arbacia pustulosa sea urchin. In thestudy of Salaque et al., sea urchins were divided into threetreatment groups and each group was injected in the coelomiccavity with either [2-14C]-acetate, L-[methyl-14C]-methionine or[3-14C]-propionate. Ten days aer injection, the sea urchinswere sacriced, and their shells and internal organs wereextracted with different solvents and the radioactivity in theirbiological materials was determined. The highest level ofradioactivity was found to be incorporated in echinochrome Afollowing [2-14C]-acetate treatment, and the radioactivity levelremained constant aer three recrystallization steps to excludecontaminants and impurities. Interestingly, the radioactivity ofthe side chain of echinochrome A was relatively low, so it wassuggested that biogenesis may proceed in two stages – rst,cyclisation of a single polyketide derived from ve acetateresidues and then the introduction of the side chain. This wasin agreement with the observation that spinochromes both withand without a two-carbon side chain (either ethyl or acetyl)frequently occur together in the same animal. Thus, Salaque
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Fig. 1 Polyketide pathway of spinochrome biogenesis.
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et al. (1967) established the de novo synthesis of echinochromeA in sea urchins and suggested a common scheme for spino-chrome biogenesis (Fig. 1). Nevertheless, the authors did notexclude the participation of microbial ora in the biosynthesisprocess.
Another argument supporting the polyketide origin of spi-nochromes relates to the meta-positions of hydroxyls in spino-chromes A and B, and that all alkylated naphthoquinones of seaurchins (as can be seen from Table 1) have a –COCH3 or –CH2–
CH3 side chain. However, no naphthoquinones having a methylside-chain that is characteristic for plant quinones bio-synthesized by other pathways (Table 1) are known. Thesefeatures seem signicant and are consistent with the polyketidehypothesis of biosynthesis formulated above.
To achieve polyketide biosynthesis in sea urchins, there isa need for specic enzymes. Polyketide synthases (PKS) area large family of multifunctional enzymes. The PKSs catalyzethe biosynthesis of different groups of compounds, which aremainly produced by bacteria, fungi, and plants. Animal PKSgenes were rst discovered in sea urchins, indicating that spi-nochrome pigments are produced by sea urchins with their ownenzymes and likely not involving endosymbionts.30
Ageenko et al. (2011)31 analyzed PKS and sulfotransferase (sult)gene expression in embryos and larvae of the sea urchin Strong-ylocentrotus intermedius at various stages of development and inspecic tissues of the adults. The authors found that addition ofshikimic acid to zygotes and embryos increased the expression ofthe PKS and sult genes. Based on this, the authors suggested thatspinochromes are generated aer a series of enzymatic, oxidative,and photochemical reactions from shikimic acid, like thatoccurring for the formation of the plant quinone chimaphilin.However, animals (Metazoa), including sea urchins, do not havea shikimate pathway,32 since proteinogenic aromatic amino acidsand other necessary products of the shikimate pathway are ob-tained in sufficient quantities from food (or from symbionts), itwould appear that animals have not had a need to evolve de novobiosynthesis of shikimate derivatives.
4. Functions of spinochromes
Despite spinochromes having been studied for quite a longtime, it is still not known what functions they perform in seaurchins. Since secondary metabolites are oen considered toplay a protective role against bacteria, fungi, amoebae in plants,insects, and large animals,33 spinochromes might be utilized bysea urchins for protection. It has been shown that sea urchinextracts and individual spinochromes exhibit antimicrobial,34
antibacterial35–38 and anti-algal39 activities. Considering thatquinonoid pigments are located in the shell of sea urchins, itseems logical that they may be intended for protection from
This journal is © The Royal Society of Chemistry 2018
various pathogens.34–39 Early suggestions that spinochromeshave a respiratory function could not be conrmed,40,41 and theparticipation of spinochromes in photoreception remains anopen question.18 The presence of pigment granules with anunknown composition in coelomic uid,42 eggs43 and embryos44
of sea urchins has also been reported. It is believed that thepigment granules of the coelomic uid, the red spherulocytes,might be involved in the immune response of sea urchins. Manystudies have shown that the number of red spherulocytessignicantly increases with stress in sea urchins, such as stresscaused by a change in the temperature of the water or itssalinity,45,46 hypoxic conditions,47 or wounding of the shell.48
The role of the pigment inclusions in the jelly coat of eggs ofsome sea urchin species and in embryos is still unclear.
5. Distribution of spinochromes
Currently there are slightly more than 1000 extant sea urchinspecies known from the oceans of the world, including theArctic and Antarctic seas.49 However, only a very small numberof investigations have been performed on spinochromes inthese species to date. A search of the literature indicates thatonly around 70 species from the class Echinoidea have beeninvestigated by marine natural product chemists. Andersonet al. (1969)7 conducted a large-scale study of the distribution ofspinochromes in the shells of almost 60 species of regular(spherical) and irregular (at and heart shaped) sea urchins.The data were summarised in a review and in a monographyreported by his co-worker Thomson.7,18 The present review willfocus on the distribution of spinochromes in sea urchins thathave not been mentioned by Anderson et al.7 and Thomson18
and highlight the differences in pigment composition that havebeen reported more recently (Table 2).
5.1 Regular sea urchins
5.1.1 Family Strongylocentrotidae. Sea urchins of thegenus Strongylocentrotus are very similar in morphology, butthey are widely distributed geographically53 and vary in spino-chrome composition.
Strongylocentrotus intermedius. The sea urchin S. intermediusis a valuable shery resource of the Sea of Japan because theiredible gonads are considered as a high value product in Asia.51
The composition of S. intermedius quinonoid pigments wasinvestigated by Utkina et al. (1976)24 who isolated spinochromesA, B, C, D and 6,60-ethylidene-bis(2,3,7-trihydroxynaphthazarin).Later Li et al. (2013)51 isolated spinochrome B from S. inter-medius using macroporous resin extraction from a crudepigment extract.
RSC Adv., 2018, 8, 32637–32650 | 32641
Table 2 Distribution of spinochromes in sea urchins
Order, family, species
The six main spinochromes
Others Reference1 2 3 4 5 6
Camarodonta; EchinometridaeEchinometra mathaei + + + + — 6
+ + + + + — 35Anthocidaris crassispina(transferred to Heliocidaris crassispina)
+ + + + — 50
Camarodonta; ParechinidaePsammechinus miliaris + + + + + Sulphate derivatives of 3 and 6, 32 28Camarodonta; StrongylocentrotidaeMesocentrotus nudus + + + + + 19 21
+ + + + + Acetylaminotrihydroxynaphthoquinone,17, 32
5
+ + + + + 32 17Strongylocentrotus droebachiensis + + + + 25, 26 25
+ + 25, 26, spinochrome dimers withmolecular masses 536 and 484
4
+ + + + 20–23, 26 and 27 23Strongylocentrotus intermedius + + + + 25 24
+ — 51Strongylocentrotus pallidus + 32 23Strongylocentrotus polyacanthus + + + + + + Dimethoxy derivative of 6, 32 23Camarodonta; ToxopneustidaeToxopneustes pileolus + + + — 52
+ + + + + + — 35Tripneustes gratilla + + + 17 35Clypeasteroida; EchinarachniidaeEchinarachnius parma + + + 23, 26, 27, 30 and 31 23Clypeasteroida; ScutellidaeScaphechinus mirabilis + + 23, 26–29, 30 and 31 15, 16
and 27Diadematoida; DiadematidaeAstropyga radiata + + + 23, 26 26Spatangoida; SchizasteridaeBrisaster latifrons + + + 26 23Stomopneustoida; GlyptocidaridaeGlyptocidaris crenularis + + + — 51
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Strongylocentrotus droebachiensis. S. droebachiensis iscommonly known as the green sea urchin because of its char-acteristic green colour. It is commonly found in northernwaters all around the world including in both the Pacic andAtlantic oceans. S. droebachiensis from the Beringsea was investigated25 and spinochromes A, C, D, and E and anhy-droethylidene-3,30-bis-(2,6,7-trihydroxynaphthazarin) (now revisedto anhydroethylidene-6,60-bis-(2,3,7-trihydroxynaphthazarin)) wereisolated from the shells and spines. Shikov et al. (2011)4 usingHPLC-DAD-MS detected spinochromes D and B, two binaph-thoquinones with a molecular mass of 484, a binaphthoquinonewith a molecular mass of 502, and an unidentied spinochromedimer with a molecular mass of 536 in S. droebachiensis from theBarents Sea.
S. droebachiensis collected from the Barents Sea, the BeringSea and in the Sea of Okhotsk had different spinochromecompositions.23 Even S. droebachiensis collected in the Sea ofOkhotsk, but from different depths, were found to vary inpigment composition.23 Samples from a greater depth con-tained echinochrome A and 7,70-anhydroethylidene-6,60-
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bis(2,3,7-trihydroxynaphthazarin) as the main compounds,along with spinochrome E, its monomethyl ether namako-chrome, spinochrome D, mirabiquinone and two echino-chrome A monomethyl ethers. In contrast, the samplescollected from shallow waters contained 7,70-anhy-droethylidene-6,60-bis(2,3,7-trihydroxynaphthazarin) as themain compound and only trace quantities of echinochrome A.23
The samples collected from shallow waters also containedethylidene-3,30-bis(2,6,7-trihydroxynaphthazarin), mir-abiquinone, spinochromes E, D, and B, and an unidentiedpigment withm/z [M�H]� of 483, which was assumed to be thesame pigment detected by Shikov et al.4 Collectively, theseresults suggest that the composition of the secondary metabo-lites of marine organisms may signicantly differ due togeographic, seasonal, gender and other variations.23
Strongylocentrotus pallidus. This species is a subtidal speciesfound at depths of up to 1600m. It inhabits rocky areas, and it isfound in the Pacic from the Kamchatka Peninsula to northernAlaska and Oregon, as well as in the North Atlantic. These seaurchins were difficult to harvest, and for a long time, they wereconfused with S. droebachiensis. Therefore, the chemical
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composition of this species was only studied recently. Onlyspinochrome E and spinamine E were identied in shell andspine extracts of S. pallidus.23
Strongylocentrotus polyacanthus. S. polyacanthus is abundantamong the Aleutian Islands, in the Bering Sea, and along thecoasts of the Kamchatka Peninsula, Korea, and Japan. Thespinochrome composition of S. polyacanthus was investigatedrecently for the rst time in samples that were collected near theKuril Islands chain.23 One S. polyacanthus sampling containedtwo main compounds, spinochrome E and echinochrome A,and in addition, three minor spinochromes, spinamine E, andspinochromes C and A were found. However, in another S.polyacanthus sampling, the main compounds found were spi-nochromes E and A, and they were present in a different ratiocompared to a previous sample. Spinochrome C was absent, butspinamine E, spinochromes D and B, and a dimethoxy deriva-tive of spinochrome E were present. Again, this indicated thatthe location of sea urchins can affect the spinochromecomposition and consequently the biological activity of extractsobtained from the sea urchins.
Mesocentrotus nudus. Sea urchins of the species M. nudusbelong to the family Strongylocentrotidae that previously wereconsidered to belong to the genus Strongylocentrotus but weretransferred to the genus Mesocentrotus based on the results ofDNA–DNA hybridization and comparative morphology.54 M.nudus is a dominant sea-urchin species in the northwest Pacicand is found on intertidal and subtidal rocky sea bottoms.55 Thequinonoid pigments of the shells and spines ofM. nudus consistmainly of spinochromes A, B, and C and echinochrome A.Although 3-acetyl-2,7-dihydroxy-6-methylnaphthazarin was iso-lated from the test and spines of M. nudus in 1977,21 it wasobtained aer vacuum sublimation and has not been detectedsince then in extracts ofM. nudus, so this pigment may not be ofnatural origin. In 2012 two new aminated quinonoid pigments(aminopentahydroxynaphthoquinone and acetylaminotrihy-droxynaphthoquinone) were found inM. nudus by using UPLC .5
Aminopentahydroxynaphthoquinone was isolated by Vasilevaet al. (2016)17 and its structure was established as 2-amino-3,5,6,7,8-pentahydroxy-1,4-naphthoquinone using 1D- and 2D-NMR and was named spinamine E.
5.1.2 Family ToxopneustidaeToxopneustes pileolus. T. pileolus, commonly known as the
ower urchin, is a widespread and commonly encounteredspecies of sea urchin in the Indo-West Pacic region. UnlikeStrongylocentrotus, it is considered highly dangerous, as it iscapable of delivering extremely painful and medically seriousstings when touched. Many studies of the sea urchin T. pileolushave been dedicated to investigating toxic compounds releasedfrom its various organs, especially the pedicellariae. Kol'tsovaand Krasovskaya (2009)52 observed only spinochrome A in thepedicellariae of this species that was collected near Nha TrangBay in the South China Sea, while spinochrome A, B and C werepresent in the shells and spines at different ratios in relation tothe colour intensity of shells. T. pileolus collected from the coralreef at Toliara, Madagascar, contained echinochrome A andspinochromes A–E.35
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Tripneustes gratilla. T. gratilla, also known as the collectorurchin, inhabits the waters of the Indo-Pacic, Hawaii, the RedSea, and the Bahamas. Early studies on this species isolatedonly spinochrome A and mompain from the shells of T. gratillafrom Hawaii,20 but recent studies on T. gratilla harvested nearMadagascar reported a different pigment pattern that con-tained spinochromes D and E and echinochrome A.35
5.1.3 Family GlyptocidaridaeGlyptocidaris crenularis. The sea urchin G. crenularis is the
only species known of the Glyptocidaris genus. It lives in thewaters of the Pacic Ocean and is distributed widely in the Seaof Japan and the Yellow Sea of China.56 Spinochrome E, spi-nochrome D and spinochrome B were tentatively identied inthe pigment extract prepared from G. crenularis.51
5.1.4 Family ParechinidaePsammechinus miliaris. P. miliaris, also known as the green
sea urchin or shore sea urchin, is found in shallow waters of theEastern Atlantic Ocean and the North Sea. The gonads of P.miliaris are eaten particularly in Mediterranean cuisine.57 LC-MS analysis of shell and spine extracts indicated the presenceof spinochromes A, B, C and E, spinamine E and echinochromeA, but also revealed the existence of other related componentswith mass spectral properties consistent with sulphated orphosphorylated spinochromes B and E.28
5.1.5 Family EchinometridaeHeliocidaris crassispina (earlier called Anthocidaris crassis-
pina). H. crassispina is a species containing edible gonads that isfound in the Pacic coastal regions around Ibaragi, and in thesouthern Japan Sea around Akita, and around Taiwan and theSoutheast coast of China.58 Kuwahara et al. (2009)50 using LC-MS, identied spinochromes A, B and C and echinochrome Ain the shells of H. crassispina, in agreement with an earlierreport.7
Echinometra mathaei. E. mathaei, the burrowing urchin, isfound in the shallow waters of the Indo-Pacic region. Itsdistribution range extends fromMadagascar on the East Africancoast to the Red Sea and to Hawaii. Themain pigments found inthis species are the spinochromes A, B and C,7 and recently thepresence of echinochrome A6 and spinochrome E35 werereported.
5.1.6 Family DiadematidaeAstropyga radiata. A. radiata, known either as the red
urchin, re urchin, false re urchin or blue-spotted urchin, is alarge species with long spines and is found in the tropicalIndo-Pacic region.49 Spinochrome E and D,echinochrome A, binaphthoquinones ethylidene-3,3-bis(2,6,7-trihydroxynaphthazarin) and 7,70-anhydroethylidene-6,60-bis-(2,3,7-trihydroxynaphthazarin) have been reported to be present inthis species.23,27
5.2 Irregular sea urchins
5.2.1 Family EchinarachniidaeEchinarachnius parma. E. parma is a species of sand dollar
native to the Northern Hemisphere. It is found in the NorthPacic, on the North American east coast as well as in Alaska,Siberia, British Columbia, and Japan, and in the Northwest
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Fig. 2 Summary of extraction, purification and identification methodsfor spinochrome pigments from sea urchin shells and spines.
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Atlantic. It inhabits isolated areas on sandy bottoms below thelow tide level and down to a depth of 1500 m.49 Identication ofPHNQ pigments from E. parma was performed for the rst timerecently.23 It was found that E. parma had three main compo-nents: spinochrome D, 7,70-anhydroethylidene-6,60-bis(2,3,7-trihydroxynaphthazarin) and echinochrome A, as well as spi-nochrome E, mirabiquinone and ethylidene-3,30-bis(2,6,7-tri-hydroxynaphthazarin), and two aminated derivatives,echinamines A and B, were detected in trace quantities.
5.2.2 Family ScutellidaeScaphechinus mirabilis. The sand dollar S. mirabilis is
distributed around the Commander Islands, on the east coast ofthe Kamchatka Peninsula, the Japanese Islands and in Peter theGreat Bay (Sea of Japan) on so sea oors.59 In the last decade,several novel spinochromes have been discovered in S. mir-abilis. Echinamines A and B, which were the rst examples ofnatural polyhydroxynaphthazarins containing a primary aminogroup, were discovered in 2005.16 Aer that spinazarin (2,3,5,8-tetrahydroxy-1,4-naphthoquinone) and ethylspinazarin(6-ethyl-2,3,5,8-tetrahydroxy-1,4-naphthoquinone) were rst isolatedfrom S. mirabilis.15 Mirabiquinone, the rst unsymmetricalbinaphthoquinone, was also discovered in this species.27
5.2.3 Family SchizasteridaeBrisaster latifrons. B. latifrons is a heart shaped urchin
occurring on sandy sea oors at a depth of up to 1820 m fromthe west coast of Central America, to the Galapagos Islands,and to the west coast of Mexico, and in the Gulf of California.60
In B. latifrons extracts, echinochrome A was found to be themain compound along with three other minor components,spinochromes E and D, and 7,70-anhydroethylidene-6,60-bis(2,3,7-trihydroxynaphthazarin) that were identiedrecently.23
From the reported data on spinochrome composition indifferent sea urchins, it can be seen that spinochrome pigmentcompositions are not always species-specic. In addition, seaurchins from one genus may have different compositions ofspinochromes, as for example in the case of the Strong-ylocentrotus sea urchins (Table 2). Nevertheless, it is possible totrace certain regularities. For example, both sand dollars fromthe order Clypeasteroida contain echinochrome A and binaph-thoquinones (23, 26 and 27), and the majority of tropical seaurchins contain echinochrome A as a main pigment.
Interestingly, in many cases recent spinochrome compositiondetermination differs from that published previously. Forexample, earlier ndings by Anderson and co-authors did notdescribe echinochrome A as being present in E. mathaei, H.crassispina, P. milliaris, T. gratilla,7 but recent reports have foundthis compound in all of these species.6,28,35,61 It was likely morechallenging to identify spinochromes earlier because of a lack ofmodern instrumentation such as LC-MS and high-resolutionNMR. It is also well known that the composition of secondarymetabolites in marine organisms may differ signicantly due togeographic, ecological, seasonal, gender and other variables.Therefore, it is very likely that differences in spinochromecompositions in sea urchins of the same species may be due tothese variables. For example, S. droebachiensis collected from theBarents Sea,4 the Bering Sea21 and the Sea of Okhotsk23 contained
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different compositions of spinochrome pigments. Even S. droe-bachiensis collected in the Sea of Okhotsk, but from differentdepths, had variations in pigment composition and content.Since several sea urchin species such as S. mirabilis and S. droe-bachiensis are harvested commercially and the pigments extrac-ted are being used in biomedical preparations, it is important totake into account geographic, environmental and other factorsand provide information on the composition of quinonoidpigments before processing raw materials.
6. Extraction, fractionation andidentification of spinochromes
Considering that sea urchin quinones most commonly occur inshells and spines in pigment granules associated with the Ca2+
and Mg2+ based mineral phase and proteins, chemical reagentsneed to be used to release the pigments from this material. Thegeneral scheme of spinochrome pigment isolation is to treat seaurchin skeleton with an acidic solution to solubilise the mineralstructure, followed by extraction of the pigments from the acidicsolution with one or more organic solvents and then fraction-ation of the crude pigment mixture by chromatography (Fig. 2).
Hydrochloric acid is commonly used for dissolving the shelland spine mineral structure and the obtained solution isexhaustively extracted with diethyl ether.4,13,35,50,62,63 The use ofother organic solvents has been reported, including chloro-form,23 ethyl acetate28 and butanol52 that have been used toextract the pigments from the acid solubilised solution. Zhouet al.5 and Li et al.51 noted that the reaction of hydrochloric acidwith sea urchin skeleton generated bubbles of carbon dioxidewhich they said affected the subsequent extraction, so they re-ported the use of an HCl–ethanol-aqueous solution with furtherfractionation of the pigment extract on NKA-9 macroporousresin. Mishchenko and co-workers16,17,23,27 used ethanol
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containing 10% H2SO4 for spinochrome extraction which formswater-insoluble CaSO4, that in contrast to the water-solubleCaCl2 produced by hydrochloric acid solubilisation, can beeasily removed by ltration or centrifugation. Followingvacuum concentration, the ethanolic extract was then parti-tioned between water and either chloroform or ethylacetate.16,17,23,27 Some investigators have used organic acids forthe release of pigments from the shells and spines. For example,Powell et al.28 suggested formic acid for spinochrome extractionbecause carbon dioxide was not generated as a result of thereaction between the acid and calcium carbonate in the shell.Further pigment mixture fractionation was performed usingsolid phase extraction (SPE) GIGA C18E units that alloweda cleaner and better fractionation, but the authors noteda reduced yield of pigments due to a low desorption rate.28
Methods of purication and identication of spinochromepigments are similar to those commonly used in naturalproduct chemistry. Usually spinochromes are separated bycolumn chromatography on acid-washed silica-gel,7,24,25,63 fol-lowed by gel-chromatography on Sephadex LH-20 62 or reversed-phase chromatography on Toyopearl HW-40.16 To obtain moresoluble forms of spinochromes for effective separation duringsubsequent chromatography, acetates, leuco-acetates andmethyl ethers of hydroxyquinones can be prepared. Based onthe assumption that spinochrome methyl ethers do not occurnaturally, Thomson et al.18 methylated partially puried frac-tions, separated them and hydrolysed the isolated individualmethyl ethers. However, as spinochrome methyl ethers havesubsequently been found in several sea urchin species,7,22,23,25
this technique should be applied with careful consideration.18
Various colour reactions have been used for the identica-tion of quinonoid pigments.18 Over the years, colour reactionsand TLC-chromatography have been replaced with modernspectroscopic methods. A signicant contribution to spectro-scopic and mass-spectrometric studies of spinochromepigments was made by a group of scientists under the super-vision of Paul Scheuer from the University of Hawaii, who used1H NMR, UV and mass-spectrometry to study a large number ofsubstituted naphthoquinones.11,64,65
The absorption spectra of spinochromes are very character-istic and consist of a combined benzenoid and quinonoid bandat l 254–290 nm, a small quinonoid band in the 310–480 nmregion and a benzenoid multi-banded absorbance centred near500 nm.65 The presence of a methoxyl substituent in a 1,4-naphthazarin molecule bathochromically shis the absorbanceband at 500 nm to 520–530 nm.23,66 The quinonoid electrontransfer band and combined benzenoid and quinonoid band inthe absorption spectrum of the aminated spinochromes areusually bathochromically shied 15–20 nm compared with thatof their hydroxylated analogues.16,17 Acetyl substituentscontribute unambiguously to the absorption spectra of spino-chromes, as for example, despite spinochromes A and C bothhaving an acetyl substituent and differing only in one hydroxylgroup, their absorption spectra are quite different.5,23
The quinonoid carbonyl frequencies of naphthazarins areuseful diagnostic aids in the determination of their structures.18
The carbonyl absorption nCO of naphthazarins occurs at 1590–
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1620 cm�1 and in the same interval C]O and C]C correlationsare displayed as a mixed band. In the IR spectra of naph-thazarins, there is a broad (2200–3400 cm�1) absorption bandcorresponding to a hydroxyls that form a strong intramolecularhydrogen bond with quinonoid carbonyls. Signals emanatingfrom b hydroxyl and amino substituents are located atfrequencies in the range of 3310–3525 cm�1.67
Moore et al. (1966)11 correlated the chemical shis in 1HNMR spectra of hydroxyl, acetyl, ethyl, methoxyl, and acetoxylsubstituents and their inuence upon each other at variousnaphthoquinone ring positions. It was established thata hydroxyl protons give singlet signals around dH 11.5–13.3.Generally, signals of b hydroxyl protons (dH 6.4–10.5) and aminogroup protons (dH 5.4–6.0) appear as broadened singlets. Thechemical shi of the free proton in the naphthoquinonenucleus is around dH 6.2–7.6. Ethyl substituents give two char-acteristic signals – a quartet of a methylene group (dH 2.6–2.8)and a triplet of a methyl group (dH 1.1–1.3). The singlet signal ofan acetyl substituent has a dH value around 2.5–2.9, and ofa methoxyl, 3.9–4.2. Signals of the ethylidene bridge thatconnects naphthazarin moieties of bi-naphthoquinones arecomprised of a quartet at dH 4.4–4.8 and a doublet at dH 1.4–1.6.Until recently 13C NMR data was not available for the majority ofspinochromes, but in last decade it has been reported fora number of these compounds. Chemical shis associated withthe nodal carbons C-9 and C-10 have dC values around 101.8–110.2. Signals of carbonyl carbons C-1 and C-4 appear at dC
169.0–182.8. Carbons attached to hydroxyls have a dC value of135.8–160.6. The chemical shi of the carbon atom substitutedwith an aliphatic chain is around dC 120.0–136.7. The methylcarbon of an ethyl substituent has a chemical shi at dC 12.0–13.5, and a methylene carbon at dC 16.2–17.2. An ethylidenebridge exhibits a signal corresponding to a methyl carbon at dC17.6–22.7 and a methine carbon at dC 23.1–27.7. The signal ofa methoxyl carbon has a chemical shi of around dC 60.9–61.6.
In the last decade, a total chemical shi assignment basedon 2D NMR experiments, specically Heteronuclear MultipleBond Correlation (HMBC), have been reported for a number ofspinochrome pigments. Interpretation of HMBC data for spi-nochromes is usually complicated because the proton signals ofb hydroxyls are broad and give no sharp peaks. To date, forseveral spinochromes a total chemical shi assignment hasbeen reported based on 2D NMR, including spinochrome D,26
echinochrome A,26 echinochrome A methyl ethers,23 echin-amines A and B,16 spinamine E,17 mirabiquinone,27 ethylidene-3,30-bis(2,6,7-trihydroxynaphthazarin) and 7,70-anhydro-ethyl-idene-6,60-bis(2,3,7-trihydroxynaphthazarin).26
Although X-ray crystallography has played a key role indetermining the structure of numerous marine natural prod-ucts for decades, there is only one report of a crystallographicstudy of sea urchin quinonoid pigments.68 This X-ray analysisexamined the molecular structure of echinochrome and thestable complex formed during the extraction and purication ofechinochrome using dioxane.68
The electron-impact-induced fragmentation characteristicfor naphthoquinones is the loss of one or two molecules ofcarbon monoxide and elimination of an acetylenic fragment
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from the quinonoid ring. If the remaining fragment is hydrox-ylated, the breakdown is accompanied by a characteristichydrogen rearrangement.69 Nowadays, the most common typeof ionization for spinochrome identication is electrospray(ESI), under such conditions PHNQ form [M � H]� ionseasily.4,5,23,28,35,51 High resolution ESI mass spectrometry andMS/MS techniques have been widely used.4,5,28,51 MS/MS fragmen-tation involves either a loss of a molecule of water (M–18), ora molecule of carbon monoxide (M–28), or loss of both of them(M–46).28
Currently, HPLC coupled with UV and mass detection isbeing used as a rapid and accurate method for the identicationof spinochrome pigments.5,6,23,35,51,61
7. Stability of spinochromes
It is important to investigate the stability of spinochromes duringisolation, separation and storage since it is critical for their func-tionality and application. However, there are only a few publica-tions available on the stability of spinochromes under differentchemical conditions. Zhou et al.70 investigated the stability ofspinochrome crude mixtures recovered from S. nudus shells. Theauthors found that pigments in aqueous solution were prone todegradation upon exposure to light and elevated temperature.Several factors such as exposure to alkaline conditions, oxidizingagents and some metal ions such as Ca2+, Zn2+, Al3+ and Fe3+ mayincrease the oxidation rate of the pigments.70,71 However, theauthors used for their experiments a centrifuged aqueous solutionof pigments, despite that most spinochromes are poorly soluble inwater. Consequently, the characteristic naphthoquinone absorp-tion band at 470 nm in the absorption spectra that they obtainedwas quite low in intensity, compared to other bands, that indicatedthe low purity of the spinochromes solutions. Hence, the conclu-sions made by the authors about the stability of spinochromesunder different conditions requires careful consideration.
Shikov et al.72 evaluated the stability of individual pigmentsfrom S. droebachiensis, including spinochrome D, an unidenti-ed spinochrome dimer and ethylidene 6,60-bis(2,3,7-trihydroxynaphthazarin). The dimeric pigments remainedstable in strongly acidied ethanol solution at pH 1.6, and wereprone to degradation with an increase in pH up to 6.0, whilespinochrome D was found to be stable up to pH 4.0. Vasilevaand co-authors compared the stability of aminated spino-chromes and their hydroxylated analogues in HEPES buffer atpH 7.5.17 Echinochrome A, echinamine A and echinamine Bwere found to be stable under these conditions, while spino-chrome E and spinamine E were prone to degradation.
8. Bioactivities of spinochromes
Even though many investigations of the bioactivities of spino-chromes have been conducted, most of these studies have beenbased on crude extracts and not on isolated spinochromes. Inthis review we will focus mainly on the bioactivities of indi-vidual pigments.
A number of investigations have reported that spinochromesare strong antioxidants that can block a number of free radical
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reactions, inhibit lipid peroxidation, and reduce and chelatemetal ions.5,6,28,50,51,73–76 The antioxidant effect in vitro of spino-chromes depends on the number and mutual positions of thechemical substituents in the molecule.75,76 Substitution ofhydroxyl groups with methoxyls in the naphthoquinone mole-cule leads to the loss of antioxidant activity.75 Recently it hasbeen shown that aminated spinochromes are more potentantioxidants than their hydroxylated analogues.16,76
Echinochrome A and the spinochromes A–E were studied fortheir antimicrobial activity by Stekhova et al.,34 who reportedthat only echinochrome A exhibited some antimicrobial effecttowards the fungi Saccharomyces carlsbergensis, Candida utilisand Trichophyton mentagrophytes, and towards Staphylococcusaureus bacteria. Unlike antioxidant activity, the antimicrobialeffect of spinochromes did not depend on the location andnumber of hydroxyl groups in the naphthoquinone molecule,while the presence of the ethyl substituent considerablyincreased the antimicrobial activity. The antimicrobial activityof trimethyl esters of echinochrome A and spinochrome B wassignicantly higher than that of their hydroxylated analogues.34
Brasseur et al.35 compared the antibacterial activity of crudeextracts and individual spinochromes from four sea urchinspecies and demonstrated that the results of bioassays differwhen using crude extracts compared to puried spinochromes.The echinochrome A/spinochrome Cmixture and spinochrome Awere active inhibitors against Shewanella oneidensis. Spino-chromes B and E had variable inhibitor activity that was depen-dent on the bacterial strain. The authors showed spinochrome Band spinochrome E had high antibacterial activity againstEscherichia coli, but they were less effective against other bacteriasuch as Bacillus subtilis and Vibrio aestuarianus. Brasseur et al.35
also showed that each isolated spinochrome signicantlyincreased the TNF-alpha production by LPS-stimulated macro-phages inducing a pro-inammatory activity, and that all isolatedspinochromes exhibited low cytotoxicity against HeLa cells.
Echinochrome A and spinochrome A appeared to be inhib-itors of tyrosine hydroxylase and dopamine-b-hydroxylase,which are targets for treatment of hypotension and otherconditions.77,78 However, the inhibition mechanism is still notwell understood. The authors suggested that this might be dueto formation of a complex through an enzyme-bound metal, ordue to the non-enzymatic oxidation of the reducing cofactor andthe resultant consumption of oxygen in the reaction mixture.
Until recently there was no information about bioactivities ofbinaphthoquinones from sea urchins except for antioxidantactivity.27 Recently, an extract of Strongylocentrotus droebachiensiscontaining the spinochromes B and D and binaphthoquinones25 and 26, was shown to exhibit a signicant anti-allergic prop-erty.3 It reduced the histamine-induced contractions of isolatedguinea pig ileum in a dose-dependent manner, had an inhibitoryeffect on a model of ocular allergic inammation, and did notshow any irritating effect in rabbits.3 Molecular docking ofinvestigated compounds into an H1R receptor demonstrated thatbothmonomer and dimer spinochromes bind successfully to thereceptor active site, but monomers t better. On the basis of thisstudy a biomedical preparation is being developed in Russia withanti-inammatory and anti-allergic effects.79
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Scheme 1 Synthesis of spinochromes as described by Singh et al.90 Reagents and conditions: (i) AlCl3–NaCl, 195 �C; (ii) MeONa, MeOH, reflux;(iii) conc. HBr, reflux.
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9. Biomedical applications ofechinochrome A
Among all the known spinochromes, echinochrome A is themost well studied compound in terms of biomedical applica-tions. The antioxidant pharmaceutical “Histochrome” wascreated on the basis of the pigment echinochrome A.73 Thisproduct was developed at the Laboratory of the Chemistry ofNatural Quinonoid Compounds, Pacic Institute of BioorganicChemistry.73 It is a water-soluble sodium salt of echinochromeA, a pentahydroxyethylnaphthoquinone, dissolved in 0.9%sodium chloride solution. It is registered as amedicinal productauthorized for use in the Russian Federation in the pharma-cotherapeutic group “antioxidant agent”. It is recommended asan antioxidant, cardioprotector and as an ophthalmic andantiarrhythmic medicine.73 Histochrome has been demon-strated to be an effective drug for the treatment of proliferativeprocesses, degeneracies, and ophthalmic hemorrhages ofvarious types.73,80,81 However, the effect of this medicine is verydifficult to explain in terms of the antioxidant properties alone.The results of experimental investigation of echinochrome Aboth in vivo and in vitro showed that this compound has farmore biological effect than that found for the Histochromeproduct, including anti-brosis, anti-diabetic, anti-allergic,anti-acetylcholinesterase, mitochondria-protective, gastro-protective and other effects.82–89 Therefore, development of newformulations based on echinochrome directed to providingtargeted and controlled release of this compound may expandthe application of this compound.
Scheme 3 Synthesis of echinamines A (30) and B (31) as described by Pok(ii) HBr-HOAc, reflux.
Scheme 2 Synthesis of spinochrome E (6) as described by Borisova anEtOH, H2O, heating; (iii) Na2S2O4, H2O; (iv) AlCl3, PhNO2, heating, then 5
This journal is © The Royal Society of Chemistry 2018
10. Synthesis
As spinochromes have been shown to exhibit a wide range ofbioactivities, several attempts have been made to developsynthesis pathways for effective production.90–99 Synthesis ofvarious spinochromes, including echinochrome A and spino-chromes A, C, D, and E, were described ve decades ago.65,90 Thenaphthoquinone skeleton of the target compound was con-structed by condensing dihydroxy-3,4-dimethoxybenzene (A)with dichloromaleic anhydride (B) in an AlCl3–NaCl melt at195 �C. Then, the Cl atoms are replaced with OMe (ii) groupsand demethylated with HBr (iii) according to the conditionsgiven in Scheme 1.
However, this synthesis pathway was not suitable forpreparative use due to cost. At present, the Anufriev group hasdeveloped pathways of preparative synthesis for almost all ofthe spinochromes. As starting compounds, commerciallyavailable 5,8-dihydroxy-1,4-naphthoquinone (naphthazarin) or2,3-dichloronaphthazarin are usually used. Thus, echino-chrome A,91 spinochrome C,92,93 spinochrome D,92 spinochromeE,94 echinamines A and B,95 spinamine E,96 and a pigment ofEchinothrix diadema containing a pyran unit, 2-hydroxy-20-methyl-20H-pyrano[3,2-b]-naphthazarin (18)97 have beensynthesized.
Recently, a more simple and effective synthesis of spino-chrome E has been described by Borisova and Anufriev.94
Preparative synthesis starts with 2,3-dichloro-6,7-diethoxynaphthazarin (C) with the simultaneous replacementof Cl atoms with hydroxyl and nitro groups followed by reduc-tion of the latter, and subsequent removal of alkoxy groups
hilo et al.95 Reagents and conditions: (i) NaN3, DMSO, 50 �C, then H2O;
d Anufriev.94 Reagents and conditions: (i) (EtO)3CH, reflux; (ii) NaNO2,% HCl, heating.
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Scheme 4 Synthesis of bis(trihydroxynaphthazarin) (23).98 Reagentsand conditions: (i) MeNH2$HCl, EtOH, heating 4 h; (ii) AlCl3, PhNO2,70 �C, 12 h.
Scheme 5 Synthesis of echinochrome A (1) as described by Pena-Cabrera and Liebeskind.99
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(Scheme 2). Shestak et al.92 have reported synthesis of spino-chromes C and D in gram quantities.
Echinamines A and B have been synthesized from chlor-omethoxynaphthazarin derivatives (D) by nucleophilic substi-tution of the chlorine atom with an azide group, followed byconversion to an amine and cleavage of the methoxy group byhydrogen bromide in acetic acid (Scheme 3).95
Pelageev et al.98 suggested a method for the preparativesynthesis of bis(trihydroxynaphthazarins) using spinochrome Ddimethyl ether (E). According to Scheme 4, compound E reactswith acetaldehyde under acidic conditions giving the tetrame-thoxy ether of bis(trihydroxynaphthazarin) (23). Demethylationof this product leads to bis(trihydroxynaphthazarin) (23) ingood yield.
A simple and efficient total synthesis of echinochrome Astarting with esters of squaric acid was developed by Pena-Cabrera and Liebeskind99 according to Scheme 5. Key inter-mediates F and G were efficiently prepared from diisopropylsquarate, a common and readily available starting material.Nucleophilic addition of aryl-lithium G to F, followed bythermal ring-expansion/cyclization of the 1,2-adduct H, gener-ated the hydroquinone I. Oxidation and full deprotection of Igave the target compound with a yield of 41%.
11. Conclusion and outlook
Although the chemistry of spinochrome pigments has beenstudied for more than 100 years, many questions still remain.For example, the fact that the functional value of spinochromesto sea urchins and their embryos is still unknown, highlightsthe need for further biochemical studies. This review aimed tosummarize all existing data on spinochrome biosynthesis andprovide an indication as to where there are still controversialopinions on this subject.
The challenges associated with the identication and struc-ture elucidation of spinochromes have nowadays been largelyovercome by using high-resolution mass spectrometry and 2D-NMR methods. However, there are still many compounds of
32648 | RSC Adv., 2018, 8, 32637–32650
the spinochrome class for which NMR signal assignment hasyet to be reported. Additionally, there is a need to develop moreefficient methods for production of puried spinochromes,either from the natural sources or by chemical synthesis.Traditional extraction of spinochromes from natural sourcesinvolving organic solvents potentially exposes personnel andthe environment to toxic compounds. The use of macroporousresin based extraction systems has the potential to reduce theamount of organic solvents used, however further investigationof the mechanism of action of these absorbents is required inrelation to the extraction yield of spinochromes obtained.
This review indicates that the structure–function relation-ship of spinochromes still requires more investigation. Despitethe growing number of investigations, a clear understanding ofthe relationship between the structure of spinochromes andtheir bioactivities is yet to be fully elucidated. To date many ofthe bioactivities reported for spinochromes have been deter-mined using crude extracts. There is a need for determinationof the biological activities of individual spinochromes and theiranalogues. This will enable exploration of opportunities tomaximize commercial and scientic use of these compounds,as illustrated with the development of the ‘Histochrome’product.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The rst author acknowledges the University of Otago forawarding a PhD scholarship to Yakun Hou.
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